Molecular mechanism of CCDC106 regulating the p53-Mdm2/MdmX signaling axis

The tumor suppressor p53 (p53) is regulated by murine double minute 2 (Mdm2) and its homologous MdmX in maintaining the basal level of p53. Overexpressed Mdm2/MdmX inhibits cellular p53 activity, which is highly relevant to cancer occurrence. Coiled-coil domain-containing protein 106 (CCDC106) has been identified as a p53-interacting partner. However, the molecular mechanism of the p53/Mdm2/MdmX/CCDC106 interactions is still elusive. Here, we show that CCDC106 functions as a signaling regulator of the p53-Mdm2/MdmX axis. We identified that CCDC106 directly interacts with the p53 transactivation domain by competing with Mdm2 and MdmX. CCDC106 overexpression downregulates the cellular level of p53 and Mdm2/MdmX, and decreased p53 reversibly downregulates the cellular level of CCDC106. Our work provides a molecular mechanism by which CCDC106 regulates the cellular levels of p53 and Mdm2/MdmX.


Bioinformatic characterization of CCDC106 protein
As its biological function has not been thoroughly investigated, we use the Simple Modular Architecture Research Tool (SMART) 1 to predict its putative domains.As shown in Fig. 1b, it contains a coiled coil domain predicted with confidence (Supplementary Table 1).However, it is also likely that CCDC106 may have potential to constitute other functional domains, such as Basic region leucin zipper (BRLZ), Parathyroid hormone (PTH), Repeats in fly CG4713, worm Y37H9A.As predicted using alphaFold2 program, the CCDC106 structure contains a long -helix flanked by two coils at the two ends of the -helix in the N-terminal region of the CCDC106 protein, while its C-terminal region forms a compact helix-rich structure.
Therefore, we arbitrarily define these two regions as the N-terminal domain (NTD) and the C-terminal domain (CTD) of CCDC106, respectively (Fig. S1).
Supplementary Fig. 1.Dissection of CCDC106 structure for expression in GST fusion protein.

Examination of p53 gene in H1299 genome
In normal human cells, the loco of the wild type p53 gene is in the 17 th chromosome with a length of 21070 bp embedded in a long promotor region and the p53 genome.
The p53 genome contains 11 exons and 10 introns (Supplementary Fig. 2).We used 18 pairs of specific primers to amplifyp53 DNA fragments from the H1299 genome using the p53 genome of the 293T cells as template.As shown in Supplementary Fig. 2b, the fragments 11, 12 and13 were not detected from the H1299 genome, while these three fragments could be amplified from the HCT116 genome (Supplementary Fig. 2c) that contains a wild type p53 gene 2 .Thus, the p53 genomic DNA in H1299 cells was missing the entire DNA sequence covering the region from exon 2 to exon 7 (Supplementary Fig. 2d & 2e).After amplified DNA fragments were evaluated by from ATCC, and NCI-H1299 p53+ cell line was constructed in our group 4,5 .
The primers used in this study were synthesized by GenScript (Nanjing, China).and its TAD domain (1-93) and DBD domain (94-312) were subcloned in a modified pET28b plasmid that its thrombin cleavage site was substituted with the Tev protease cleavage site 6 .
To purify His-tagged protein, cells were harvested by centrifugation at 5,000 ×g for 30 min, resuspended in a buffer containing 10 mM Tris-HCl, 40 mM NaCl, 2 mM β-mercaptoethanol, 2 mM imidazole, pH 8.0 (Buffer A), and lysed by sonication and homogenization, followed by spinning at 18,000 ×g for 30 min.The supernatant was loaded onto a 5 mL Ni-NTA agarose column (Qiagen, USA) and His-tagged protein was competitively eluted using a gradient of Buffer A mixed with Buffer B containing 10 mM Tris-HCl, 40 mM NaCl, 2 mM β-mercaptoethanol, 300 mM imidazole, pH 8.0.
To purify GST-tagged protein, cells were harvested by centrifugation at 5,000 ×g for 30 min, resuspended in 1x PBS buffer and lysed by sonication and homogenization, followed by spinning at 18,000 ×g for 30 min.The supernatant was loaded onto a 5 mL GST agarose column (GE, USA) and GST-tagged protein was competitively eluted using 1x PBS buffer containing10 mM glutathione.The eluate was desalted with a 1xPBS buffer.All purified protein samples were freshly frozen in liquid nitrogen, and kept at -80 C.

DNA sequencing of p53 genomic gene in H1299 cells
The genomic DNA of H1299 and HCT116 cells was extracted with the cell genome extraction kit, and the 53 genome was divided into 18 fragments according to the genome data on NCBI (Genebank ID: 7157).PCR was used to identify whether each fragment existed in H1299, and HCT116 was used as a control.PCR was carried out with Taq DNA polymerase in 50 μL reaction mixture, which contained 25 μL of 2 × Taq mix, 2 nM primers and 10 ng template DNA.
The total RNA of H1299 cells was extracted using the RNA extraction kit, and immediately reverse transcription PCR (RT-PCR) was performed to obtain the cDNA library of H1299, and then each putative fragment of p53 gene were amplified with designed primers (Supplementary Table 3).PCR products were evaluated by agarose gel electrophoresis.The fragments obtained by the above PCR were purified and ligated with TOPO-Blunt vector.The ligation mixture was transformed into E. coli DH5α competent cells for selecting correct colonies.After cultured for 12 hours, a single colony was picked and identified using colony PCR with primers M13-F: 5'-TGTAAAACGACGGCCAGT-3' and M13-R: 5'-CAGGAAACAGCTATGACC-3'.
PCP products were directly used for DNA sequencing.
. Primers for sequencing p53 genomic DNA and cDNAs